This invention relates generally to extraction systems and methods for separating analytes from streams containing other constituents by differential transport principles such as diffusion and applied fields, providing an improved method involving the use of absorbents or adsorbents in the extraction stream. The devices and methods of this invention can be used for diagnostic and therapeutic/treatment purposes.
Field flow fractionation devices involve particle size separation using a single inlet stream. See, e.g., Giddings, J. C., U.S. Pat. No. 3,449,938, Jun. 17, 1969, xe2x80x9cMethod for Separating and Detecting Fluid Materialsxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,147,621, Apr. 3, 1979, xe2x80x9cMethod and Apparatus for Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,214,981, Jul. 29, 1980, xe2x80x9cSteric Field-Flow Fractionationxe2x80x9d; Giddings, J. C. et al., U.S. Pat. No. 4,250,026, Feb. 10, 1981, xe2x80x9cContinuous Steric FFF Device for The Size Separation of Particlesxe2x80x9d; Giddings, J. C. et al. (1983), xe2x80x9cOutlet Stream Splitting for Sample Concentration in Field-Flow Fractionation,xe2x80x9d Separation Science and Technology 18:293-306; Giddings, J. C. (1985), xe2x80x9cOptimized Field-Flow Fractionation System Based on Dual Stream Splitters,xe2x80x9d Anal. Chem. 57:945-947; Giddings, J. C., U.S. Pat. No. 4,830,756, May 16, 1989, xe2x80x9cHigh Speed Separation of Ultra-High Molecular Weight Polymers by Hyperlayer Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,141,651, Aug. 25, 1992, xe2x80x9cPinched Channel Inlet System for Reduced Relaxation Effects and Stopless Flow Injection in Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,156,039, Oct. 20, 1992, xe2x80x9cProcedure for Determining the Size and Size Distribution of Particles Using Sedimentation Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,193,688, Mar. 16, 1993, xe2x80x9cMethod and Apparatus for Hydrodynamic Relaxation and Sample Concentration in Field-Flow Fraction Using Permeable Wall Elementsxe2x80x9d; Caldwell, K. D. et al., U.S. Pat. No. 5,240,618, Aug. 31, 1993, xe2x80x9cElectrical Field-Flow Fractionation Using Redox Couple Added to Carrier Fluidxe2x80x9d; Giddings, J. C. (1993), xe2x80x9cField-Flow Fractionation: Analysis of Macromolecular, Colloidal and Particulate Materials,xe2x80x9d Science 260:1456-1465; Wada, Y. et al., U.S. Pat. No. 5,465,849, Nov. 14, 1995, xe2x80x9cColumn and Method for Separating Particles in Accordance with Their Magnetic Susceptibilityxe2x80x9d; Yve, V. et al. (1994), xe2x80x9cMiniature Field-Flow Fractionation Systems for Analysis of Blood Cells,xe2x80x9d Clin. Chem. 40:1810-1814; Afromowitz, M. A. and Samaras, J. E. (1989), xe2x80x9cPinch Field Flow Fractionation Using Flow Injection Techniques,xe2x80x9d Separation Science and Technology 24(5 and 6):325-339.
Thin-channel split flow fractionation (SPLITT) technology also provides particle separation in a separation cell having a thin channel. A field force is exerted in a direction perpendicular to the flow direction. Particles travel from a particle-containing stream across a transport stream to a particle-free stream. The device for operating the process is generally fabricated from glass plates with teflon sheets used as spacers to form the channels. The channel depth can therefore be no smaller than the spacers, which are generally about 100 to 120 xcexcm thick. See, e.g., Giddings, J. C., U.S. Pat. No. 4,737,268, Apr. 12, 1988, xe2x80x9cThin Channel Split Flow Continuous Equilibrium Process and Apparatus for Particle Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,894,146, Jan. 16, 1990, xe2x80x9cThin Channel Split Flow Process and Apparatus for Particle Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,093,426, Aug. 13, 1991, xe2x80x9cProcess for Continuous Particle and Polymer Separation in Split-Flow Thin Cells Using Flow-Dependent Lift Forcesxe2x80x9d; Williams, P. S. et al. (1992), xe2x80x9cContinuous SPLITT Fractionation Based on a Diffusion Mechanism,xe2x80x9d Ind. Eng. Chem. Res. 31:2172-2181; and Levin, S. and Tawil, G. (1993), xe2x80x9cAnalytical SPLITT Fractionation in the Diffusion Mode Operating as a Dialysis-like System Devoid of Membrane. Application to Drug-Carrying Liposomes,xe2x80x9d Anal. Chem. 65:2254-2261.
The object of this invention is to provide an improved extraction system utilizing differential transport principles in which the analyte can be extracted, detected and quantified. A further object of this invention is to provide an improved extraction system for purification and treatment of fluids, including bodily fluids such as blood.
All publications, patents and patent applications referred to herein are incorporated in their entirety by reference.
Differential extraction devices as described above allow desired particles to move from a sample stream into an extraction stream running in parallel laminar flow with the extraction stream. A simple embodiment of such systems uses a concentration gradient across the streams so that desired particles diffuse from the sample stream into the extraction stream. Other gradients and forces can also be used, e.g., magnetic, electrical, gravitational, dielectrical, sedimentation, shear, centrifugal force, temperature, pressure, and cross-flow gradients.
An improvement in the above processes provided herein is the addition of a sequestering material to the extraction stream.
The invention provides an extraction device for extracting desired particles from a sample stream containing said desired particles, said device comprising:
a. a sample stream inlet;
b. an extraction stream inlet;
c. an extraction channel in fluid communication with said sample stream inlet and said extraction stream inlet for receiving a sample stream from said sample stream inlet in adjacent laminar flow with an extraction stream from said extraction stream inlet;
d. a sequestering material within said extraction channel for capturing desired particles in said extraction stream;
e. a by-product stream outlet in fluid communication with said extraction channel for receiving a by-product stream comprising at least a portion of said sample stream from which desired particles have been extracted;
f. a product outlet in fluid communication with said extraction channel for receiving a product comprising said sequestering material and at least a portion of said desired particles.
A sequestering material is a material which captures, e.g., by adsorbing, binding or sticking to the desired particles, or by absorbing them. Enzymes, antibodies, antigens and other ligands for desired particles are known to the art and are useful sequestering materials for this invention. Any ligand known to the art for a desired particle may be used as a sequestering material. Such ligands may be added to the extraction stream xe2x80x9cas-isxe2x80x9d or may be immobilized on substrates such as polymeric beads, high molecular weight polymers, or other materials known to the art. xe2x80x9cHigh molecular weight polymersxe2x80x9d refers to those polymers of sufficient molecular weight that they do not substantially diffuse into the sample stream during their transit through the device. Examples of high molecular weight polymers include but are not limited to high molecular weight dextrans, high molecular weight polypeptides, and high molecular weight nucleic acids. The sequestering material may also be an absorbent material such as activated charcoal, or porous polymers. Absorbents or adsorbents may be either specific to a particular particle type, such as an antibody, or nonspecific, such as activated charcoal.
The sequestering material is preferably substantially non-diffusing, i.e., should diffuse sufficiently slowly that it does not cross from the extraction stream into the sample stream to any significant degree, so that it does not become detectable in the by-product stream, or does not interfere with analysis of analytes in the by-product stream.
The sequestering material captures the desired particles by preventing them from exiting the device with the exiting by-product stream. The desired particles may be loosely bound to the sequestering material, so long as the sequestering material retains the particles long enough to prevent them from exiting with the by-product stream. The desired particles may be reversibly bound to or captured by the sequestering material so that they can be removed for further analysis or to allow re-use of the sequestering material.
Differential extraction devices fabricated on the microscale provide numerous advantages over the larger devices discussed above. Such microfabricated devices are described in application Ser. No. 08/663,916 filed Jun. 14, 1996, which is specifically incorporated herein in its entirety by reference along with all references incorporated therein by reference. Definitions of terms used in that application as applied to the microscale structures disclosed therein are applicable herein to macroscale structures as well as microscale structures. xe2x80x9cMacroscale structuresxe2x80x9d are defined herein as structures larger than microscale structures but still small enough to permit laminar flow.
The desired particles in this invention may be analytes or they may be substances that interfere with analytes. They may also be particles desired to be recovered and used for another purpose, or toxins, such as poisons or metabolites in a patient""s blood. For example, this invention can be used to detoxify blood, e.g., remove toxic metals from blood, or to detoxify other bodily fluids, e.g., to perform hemodialysis. This invention can be used in waste-water treatment, e.g., to remove impurities from water. Alternatively, this invention can be used to remove a drug or other product produced by microorganisms, e.g., bacterial cells, in a fermentation reactor without damaging the microorganisms. Such treatments can be performed in a continuous fashion.
This invention also provides a method for extraction of at least a portion of desired particles from a sample stream comprising said desired particles, comprising the steps of:
a. introducing the sample stream into the sample stream inlet of an extraction device as described above;
b. introducing an extraction stream into the extraction channel of said extraction device; and
c. introducing into said extraction channel a sequestering material for capturing the desired particles such that the desired particles are captured by the sequestering material, and such that said extraction stream, comprising said sequestering material and at least a portion of said desired particle, exits said device as a product stream, and such that said sample stream from which desired particles have been extracted, exits said device as a by-product stream.
The device and method of the present invention provide a means for performing affinity chromatography. As is understood by those in the art, affinity chromatography refers to a method of purifying or isolating desired substances and generally involves covalently attaching a specific ligand to an insoluble inert support. In affinity chromatography the ligand must have a high affinity for the desired substance, so that on passage in solution down a column the desired substance is preferentially retained by the ligand.
The present invention provides a device and method for performing affinity chromatography, however with at least one particular advantage. The extraction of a desired substance (particles) can be performed in a continuous fashion. The sample streams and extraction streams of the present invention can be run continuously through the device. Art-known affinity chromatography involves multiple steps, e.g., loading the ligand onto the inert material, flushing the column, loading the sample, rinsing, then rinsing again to release the desired substance, with product loss occurring at each step usually. In the device of the present invention, for example, a virus can be extracted from whole blood by 1) introducing an extraction stream comprising an antibody to the virus immobilized on beads and a sample stream of whole blood into the device and 2) after transfer of the virus particles to the extraction stream, releasing them by changing the pH of the solution. The beads can be chosen, for example, so that they will fall to the bottom of the channel or they can be magnetic and therefore pulled to one side of the channel with a magnet.
The sequestering material can be present in the extraction stream prior to the extraction stream""s being introduced into the extraction channel. Alternatively, the sequestering material can be added to the extraction stream by suspending or dissolving the sequestering material in a liquid which is introduced into the extraction stream, which is already in the extraction channel, via the extraction stream inlet.
The extraction system of this invention in simplest concept is illustrated by a diffusion extraction device comprising microchannels in the shape of an xe2x80x9cHxe2x80x9d. A mixture of particles suspended in a sample stream enters the extraction channel (the crossbar of the xe2x80x9cHxe2x80x9d) from one of the arms, e.g., the top left, and an extraction stream (a dilution stream) enters from the bottom left. The two streams flow together in the extraction channel; however, due to the small size of the channels, the flow is laminar and the streams do not mix. The sample stream exits as by-product stream at the upper right and the extraction stream exits as product stream from the lower right. While the streams are in adjacent laminar flow in the extraction channel, particles having a greater diffusion coefficient (smaller particles such as albumin, sugars and small ions) have time to diffuse into the extraction stream, while the larger particles (e.g., blood cells) remain in the sample stream. Particles in the exiting extraction stream (now called the product stream) may be analyzed without interference from the larger particles.
In this patent application, the flow direction of a channel is called its length (L). The channel dimension in the direction of particle transport at right angles to the length (L) is called its depth (d). The third channel dimension at right angles to both the length and depth is called its width (w). The depth (d) is therefore perpendicular to the plane of interface of the sample and extraction streams. Table 1 lists other abbreviations used herein.
The length of the extraction channel and the extraction channel flow velocity are key parameters determining the amount of time the particles have to diffuse into the extraction stream. The sequestering material provides for increased diffusion of the desired particles by decreasing the effective concentration of the desired particles in the extraction stream. That is, the sequestering material effects a shift in the equilibrium (in a positive direction) of diffusion of the desired particles into the extraction stream.
The particles in the case described above are differentially transported from the sample stream to the extraction stream using diffusion as the transport mechanism. Other means for effecting differential transport of the desired particles can also be used. The term xe2x80x9cdifferential transportxe2x80x9d means that a portion of the desired particles are transported from the sample stream into the extraction stream to the substantial exclusion of the undesired particles. For example, magnetic, electrical or other forces can be applied across the extraction stream, temperature gradients can be used, or absorbent or adsorbent materials such as antibodies can be added to the extraction stream to capture the desired particles.
The sample stream and extraction stream inlets and the byproduct stream and product stream outlets may comprise channels, reservoirs, ports, or other containers. The sample stream inlet is designed to receive a sample stream containing xe2x80x9cdesired particles,xe2x80x9d e.g., particles it is desired to extract so that their presence may be detected. The sample stream also includes other particles which are not extracted, termed xe2x80x9cundesired particlesxe2x80x9d herein. These undesired particles include particles which might interfere with the detection of the desired particles. In a preferred embodiment, the sample stream comprises whole blood. The desired particles may be albumin or other blood plasma components, and the undesired particles may be blood cells. The device is especially useful for obtaining cell-free plasma components from whole blood. Other fluids for which the present invention is useful include solutions or suspensions of DNA fragments of different lengths, proteins of varying sizes, or heterogeneous chemical reaction mixtures. Sample streams useful in the practice of this invention include fermentation broths, raw sewage, liquefied food samples, soil samples and biological fluids such as sputum, urine, and cerebral spinal fluid.
The term xe2x80x9cparticlesxe2x80x9d refers to molecules; cells; macromolecules such as proteins, nucleic acids and complex carbohydrates; small molecules comprised of one to several atoms; and ions. The particles may be suspended or dissolved in the stream. The term xe2x80x9cstreamxe2x80x9d refers to a carrier fluid such as water or other liquid, air or other gas, containing desired and/or undesired particles. The term xe2x80x9cparticlesxe2x80x9d as used herein does not include the molecules of the carrier stream.
The term xe2x80x9cextractionxe2x80x9d refers to the transfer of at least a portion, i.e., a detectable portion, of desired particles from the sample stream to the extraction stream, to the substantial exclusion of undesired particles. It is recognized that undesired particles may be transported into the extraction stream, particularly those that diffuse faster than the desired particles; however, the presence of such undesired particles will be minimized such that they do not interfere with detection or subsequent processing of the streams containing the desired particles. The transfer of undesired particles from the sample stream to the extraction stream can be minimized by pre-loading the extraction stream with such undesired particles. Pre-loading the extraction stream with undesired particles may be preferable in embodiments wherein the by-product stream is of interest, e.g., for further use or analysis. For example, if blood is to be returned to a patient""s body, the extraction stream preferably contains the appropriate concentrations of electrolytes, as will be understood by those of skill in the art. The sequestering material increases the efficiency of separation of the desired particles from the sample by decreasing the effective concentration of the desired particles in the extraction stream.
The term xe2x80x9cextraction efficiencyxe2x80x9d refers to the percentage of desired particles in the sample which are transferred to the extraction stream and exit in the product stream. Extraction efficiency can be increased by using a sequestering material.
The term xe2x80x9claminar flowxe2x80x9d of two streams means stable, side-by-side, non-recirculating, flow of two streams without mixing. There are no zones of re-circulation, and turbulence is negligible. As is known to the art, the Reynolds number of a flow is the ratio of inertial forces to viscous forces. For flow through a duct, the Reynolds number is calculated using the equation Re=xcfx81d({overscore (V)}/xcexc) where Re is the Reynolds number, xcfx81 is the mass density of the fluid, d is a typical cross-sectional dimension of the duct depending on the shape of the duct, {overscore (V)} is the mean velocity over the duct cross-section and xcexc is the viscosity.
As the Reynolds number is reduced, flow patterns depend more on viscous effects and less on inertial effects. Below a certain Reynolds number (based on lumen size for a system of channels with bends and lumen size changes), inertial effects are insufficient to cause phenomena indicative of their significant presence such as laminar recirculation zones and turbulent flow. Therefore, non-turbulent, laminar non-recirculating flow occurs in the extraction devices discussed herein. In such devices minimal dispersive mixing occurs as a result of the viscous flow velocity profiles present within any laminar viscous flow. This allows two laminar non-recirculating fluid streams to flow down an extraction channel for the purpose of desired particle extraction from one stream to the other.
The streams may be separated at the end of the conduit at any arbitrary location by precise regulation of the exit flow rate of the outlets, something which is not possible at higher Reynolds numbers not satisfying the non-recirculating and non-turbulent criteria.
The extraction stream inlet is designed to receive an extraction stream capable of accepting desired particles when in laminar flow contact with the sample stream. The extraction stream can be any fluid capable of accepting particles being transported from the sample stream. The extraction stream contains sequestering material which binds desired particles which have been transported from the sample stream to the extraction stream. Preferred extraction streams are water and isotonic solutions such as physiological saline. Other useful extraction streams comprise organic solvents such as acetone, isopropyl alcohol, supercritical carbon dioxide or ethanol. Air and other gases may also be used as sample and extraction stream carriers.
The by-product stream comprises at least a portion of said sample stream from which desired particles have been extracted, and may or may not, as discussed below, include a fraction of the extraction stream into which desired particles have been conveyed from the sample stream. The sequestering material effects greater extraction of the desired particles from the sample, thereby yielding a more pure by-product stream. If an excess of sequestering material is used and it has a high binding constant for the desired particles, then essentially all of the desired particles in the sample stream can be extracted from the sample stream upon treatment of the sample just once, depending on flow rate and extraction channel length. Without the sequestering material and assuming equal flow rates of sample and extraction fluids, the equilibrium concentration of the desired particles is 50% in the extraction stream. That is, at most, only 50% of the desired particles diffuse into the extraction stream. Therefore, without the sequestering material the sample has to be treated at least five times to remove 97% of the desired particles from the sample.
The by-product stream outlet is designed to conduct the by-product stream (composed of the sample stream and perhaps a portion of the extraction stream) that is removed from the extraction channel to disposal, recycle, or other system component, for further processing.
The product stream comprises at least a portion of said desired particles and the sequestering material. The product stream outlet, which as stated above, may comprise a product stream channel, is designed to conduct the product stream containing a detectable quantity of desired particles to a detection or further processing area or system component. A sufficient quantity of the extraction stream must be present in the product stream, comprising a sufficient quantity of desired particles, such that the presence of the desired particles is detectable in the product stream by means known to the art.
The product stream may be conducted to a reservoir chamber, or other device where it may be further treated, e.g., by separating the sequestering material from the desired particles, mixing, separating, analyzing, heating or otherwise processing, for example as disclosed in Wilding, P., et al. U.S. Pat. No. 5,304,487 issued Apr. 19, 1994, incorporated herein by reference. The by-product stream may also be conducted to a reservoir chamber or other container or apparatus for further treatment.
The devices of this invention may be xe2x80x9cmicrofabricated,xe2x80x9d which refers to devices capable of being fabricated on silicon wafers readily available to those practicing the art of silicon microfabrication and having the feature sizes and geometries producible by such methods as LIGA, thermoplastic micropattern transfer, resin based microcasting, micromolding in capillaries (MIMIC), wet isotropic and anisotropic etching, laser assisted chemical etching (LACE), and reactive ion etching (RIE), or other techniques known within the art of microfabrication. In the case of silicon microfabrication, larger wafers will accommodate a plurality of the devices of this invention in a plurality of configurations. A few standard wafer sizes are 3xe2x80x3, 4xe2x80x3, 6xe2x80x3, and 8xe2x80x3. Application of the principles presented herein using new and emerging microfabrication methods is within the scope and intent of the claims hereof.
In a preferred embodiment, called the xe2x80x9cH-filter devicexe2x80x9d herein, the inlet and outlet channels are between about 2 to 3 times the maximum-sized stream particulate diameter and about 100 micrometers in width and between about 2 to 3 times the diameter of the maximum-sized particles and less than about 100 micrometers in depth, and the extraction channel is between about 2 to 3 times the diameter of the maximum-sized particles and about 2/3 the wafer thickness in width, between about 2 to 3 times the diameter of the maximum-sized particles and less than about 100micrometers in depth, and between about 4 and about 10 times the diameter of the maximum-sized particles and less than or equal to 5 mm long.
In a second embodiment in which the particle transport direction is rotated 90 degrees from that of the xe2x80x9cH-filter devicexe2x80x9d design, called the xe2x80x9cflat extraction devicexe2x80x9d or xe2x80x9cflat filter devicexe2x80x9d herein, the inlet channels have a width equal to the extraction channel width at the entrance to the extraction channel of preferably between 2 and 3 particle diameters and about 500 micrometers, and the extraction channel is preferably between about 2 and 3 times the diameter of maximum-sized particles and less than or equal to 5 mm in width, between about 2 and 3 times the diameter of the maximum-sized particles and less than about 100 micrometers in depth, and at least about 4 times the diameter of the maximum-sized particles in length.
The extraction channel receives the inflow of the sample and extraction streams from the sample and extraction stream inlets and conducts these streams in adjacent laminar flow for a distance sufficient to allow extraction of the desired particles into the extraction stream. The length of the extraction channel can be increased by forming it in a convoluted geometry, e.g., serpentine (set of xe2x80x9chairpinxe2x80x9d turns) or coiled, as are the flow channels disclosed in Weigl et al., U.S. patent application Ser. No. 08/829,679, filed Mar. 31, 1997 and PCT Application No. PCT/US97/05245, filed Mar. 31, 1997, which are incorporated herein by reference.
The width and depth of the extraction stream channel and product outlet channels must be large enough to allow passage of the desired particles, the sequestering material, and any complex of the desired particles with the sequestering material.
If the width dimension is in the wafer thickness direction, as it is in the H-filter device embodiment, then for the silicon microfabricated embodiments of the microscale extraction devices of the present invention, the widths of the sample, extraction, product, and by-product channels, inlets and outlets are less than the silicon wafer thickness, i.e., about 300 micrometers. Alternatively, if the device is made from other materials, preferably moldable materials such as plastic, or in the xe2x80x9cflat extraction devicexe2x80x9d embodiment, then there is no theoretical maximum limit to the width. Widths up to 0.5 meter, 1 meter, and even greater are contemplated. The width has no theoretical maximum limit provided that one can control the delivery of fluids (sample stream and extraction stream) into the device, e.g., the flow rate of each fluid can be controlled across the width of the channel. The dimensions of the extraction channel are chosen to maintain laminar flow and uniform flow rate, e.g., no turbulence or build up of particles on channel walls.
If the depth dimension is in the wafer thickness direction, as it is in the xe2x80x9cflat filterxe2x80x9d embodiment, then for silicon microfabricated embodiments of the microscale extraction devices of the present invention, the depth of the sample, extraction, product, and by-product channels, inlets and exits is less than the silicon wafer thickness, i.e., about 300 micrometers. Preferably, for microfabricated devices, the depth, particularly of the extraction channel, is less than about 200 micrometers, and more preferably less than about 100 micrometers.
Some fields known to the art which may be used for differential transport of the particles in the devices of this invention are those produced by:
Sedimentation
Electrical potential
Temperature gradients
Cross Flow
Dielectrical gradients
Shear forces
Magnetic forces
Concentration gradients
Means for producing such fields are known to the art.
Because of the small size of the diffusion direction (depth) of the channels described herein, differential transport of desired particles by diffusion or other means occurs extremely rapidly, e.g., within less than about 300 seconds, and if desired, less than about one second. The presence of the sequestering material in the extraction stream provides for increased desired particle transport by lowering the effective concentration of the desired particles in the extraction stream, maximizing the effective concentration difference between the sample stream and the extraction stream. This maximizes the net transfer along the depth (diffusion dimension) of the extraction channel, thus providing rapid separation of desired particles from the sample.
The sample and extraction streams may have different properties, e.g., viscosities, densities, surface energies, homogeneities, chemical compositions and the like, which may affect the differential transport rates. System parameters may need to be adjusted and optimized to take account of these differing properties, as will be apparent to, and can be done without undue experimentation by, those skilled in the art.
The sample and extraction streams are kept in contact in the extraction channel for a period of time sufficient to allow at least an analyzable quantity, and preferably a major portion, of desired particles to be transported into the extraction stream. The flow rate of the product stream from the device may be between about 0.001 picoliter/sec and about 10 ml/sec or more in devices with large widths, e.g., greater than about 50 xcexcm. For example, an optimal flow rate for the product stream can be about 200 nanoliters/sec. As is known in the art, even the very small amounts of analytes present in such small product streams may be detected by spectroscopic and other means.
The average flow velocity, {overscore (V)}, is chosen to fit the following relationship:       V    _     less than       f    ⁢          DL              d        2            
where f is a time factor (proportionality constant) related to how long the two streams must be in contact with each other in order for a certain percentage of desired particles to be transferred from the sample stream to the extraction stream.
The volumetric flow rate (Q) per unit width (w) is thus limited to be less than f(DL)/d: Q={overscore (V)}wd, Q/w={overscore (V)}d       Q    w     less than       f    ⁢          DL      d      
It may be convenient for calculation purposes to choose f=1, and calculate the maximum flow rate per unit width based thereon. For example, for biotin (with diffusion coefficient, D=500 xcexcm2/sec in a channel of length (L)=1 cm and depth (diffusion dimension) (d)=10 xcexcm, the maximum flow rate per unit width is approximately 500 picoliters/sec per xcexcm of width.
From the above the following relationship can be derived:
d2/D=2t
which means that a molecule will diffuse across distance d (the depth of the channel) in an average time of 2t.
A xe2x80x9cmajor portionxe2x80x9d of the desired particles is more than 50% of said particles present in the sample stream.
The sequestering material enhances the efficiency of the extraction process, allowing for greater than 50% extraction (the maximum extraction obtained with equal volumes of the sample and extraction stream, but without sequestering material or other differential transport forces, e.g., magnetic and electircal fields). Preferably, the sequestering material allows for extraction of greater than about 50% to about 80% of the desired particles. More preferably, the sequestering material allows for extraction of about 75% to about 95% of the desired particles. Most preferably, the sequestering material allows for extraction of about 85% to about 100% of the desired particles.
Successful operation of the invention described herein requires precise control of volume flow rates on three of the four channels of the device (i.e., sample, extraction, product, and by-product streams). The fourth channel need not and should not be regulated, as leaving this channel unregulated will allow the device to accommodate unpredictable changes in volume of the sample because of xcex94V of mixing of the sample and extraction streams. Means for achieving precisely regulated flow rates are known to the art.
To aid in controlling the size of particles being transported to the product stream in a diffusion-based extraction system of this invention, and reduce the appearance of larger particles in the product stream, a fluid barrier may be created in the extraction channel. Such a fluid barrier is present when the extraction stream is present in sufficient volume to cause a portion of the extraction stream to flow through the by-product exit with the exiting by-product stream, as illustrated in FIG. 3. Smaller particles diffusing into the extraction stream must cross the width of this fluid barrier before being able to exit with the product stream. Such fluid barriers formed on a larger scale are discussed in Williams P. S., et al. (1992), xe2x80x9cContinuous SPLITT Fractionation Based on a Diffusion Mechanism,xe2x80x9d Ind. Eng. Chem. Res. 2172-2181, incorporated herein by reference.
By controlling the flow rate of the sample and extraction streams, the ratio of volume from each that enters the extraction channel can be controlled. The volume ratio of the sample stream and the extraction stream can also be set by the geometry of the outlet and inlet channels for a fixed delivery pressure on the sample and extraction streams. The volume flow rate of the product and by-product streams may also be controlled by manipulating the product and by-product stream pressures or by using arbitrary port (inlet) pressures and altering the flow resistance of the inlets. Whatever the control mode, the inlet and outlet channels must satisfy the criteria for minimum channel dimensions based on the size of the particulate to be processed as described herein. If the volume of the extraction stream entering the extraction channel is greater than the volume of the sample stream, and the two exit streams are identical, a fluid barrier is formed. If the volume flow rate of the product stream is too small to accommodate the entire volume flow of the extraction stream then a fluid barrier will also be formed.
Extraction devices of this invention may comprise means for controlling the volume of extraction stream in the extraction channel with respect to the volume of the sample stream, which means include a product stream outlet smaller than required to allow the entire extraction stream to exit coupled with a by-product stream outlet large enough to handle the excess extraction stream. Extraction devices of this invention may comprise multiple product stream outlets so that product streams comprising different types of desired particles may be recovered.
The devices of this invention may be utilized as a sample pretreatment system for an analytical system including sensing means for detecting desired particles in the product stream. Such means include means for mixing the product stream with an indicator stream which interacts with the desired particles so as to allow them to be detected by sensing means known to the art, including optical means, such as optical spectroscopic equipment, and other means such as absorption spectroscopic equipment or means for detecting fluorescence, chemical indicators which change color or other properties when exposed to the desired particles of analyte, immunological means, electrical means, e.g., electrodes inserted into the device, electrochemical means, radioactive means, or virtually any microanalytical technique known to the art including magnetic resonance equipment or other means known to the art to detect the presence of analyte particles such as ions, molecules, polymers, viruses, DNA sequences, antigens, microorganisms, or other factors. Preferably, optical or fluorescent means are used, and antibodies, DNA sequences and the like are attached to fluorescent markers. Indicators and microfabricated mixing means, as well as detection and sensing means are described in U.S. application Ser. No. 08/625,808 incorporated herein by reference.
In one embodiment of this invention the differential extraction device described above is integrated into an analytical system comprising means for further processing the product and/or by-product streams, such as diffusion-based mixing devices for mixing the product stream with an indicator substance (e.g., as described in U.S. application Ser. No. 08/625,808 incorporated herein by reference), and detection chambers wherein the presence of desired analyte particles may be detected. These additional processing means are preferably incorporated with the differential extraction device in a xe2x80x9clab-on-a-chipxe2x80x9d, fabricated on a standard silicon wafer. The system may comprise quantitation means for determining the concentration of the analyte particles (desired or undesired particles) in the product and/or by-product stream and/or determining the concentration of the analyte particles in the sample stream. Such means include spectroscopic equipment, potentiometric, amperometric, and dielectric relaxation equipment. Concentration determinations can be made by calculation or calibration by means known to the art and disclosed herein.
In another embodiment of this invention used for purification of fluids, e.g., waste-water treatment, hemodialysis, blood detoxification, large volumes, e.g., about 10 ml/sec of sample stream may be treated. In this embodiment, preferably the width of the extraction channel is large, e.g., up to about one meter, although as discussed above, there in no fixed theoretical maximum for the channel width. For treatment of large fluid volumes, devices of this invention, including microfabricated devices, may be connected in parallel, and optionally also in series.
It may be preferable to pre-treat the device, i.e., pre-coat the internal walls of the device to enhance performance, as will be illustrated in the Examples below. The walls can be coated with the sequestering material to be used, before the device is used to effect separation of the desired particles. Without wishing to be bound to any particular theory, it is believed that pre-coating the walls with the sequestering material prevents further adherence of sequestering material to the walls when the sample and sequestering material are later introduced into the device. Alternatively, the internal walls of the device can be pre-coated to effect surface passivation with hydrophilic coating materials, which are commercially available and include, but are not limited to, albumins (e.g., bovine serum albumin, lact albumin and human serum albumin), and art-known silanizing reagents, preferably polyethyleneglycol silanes.
As will be appreciated by those skilled in the art, numerous substitutions may be made for the components and steps disclosed herein, and the invention is not limited to specific embodiments discussed.